Koch Lab:Protocols/Microsphere-DNA tethering/Glass, dig, biotin, microsphere, 4kb DNA
References
Materials
Sample chamber
You can use more sophisticated sample chambers, obviously, but our most common method remains the ultra-low-tech "double-stick tape" method. For this you need:
- #1 coverglass (or whatever your microscopy requires). 60 x 24 mm is convenient. You can get Corning from Fisher.
- regular glass slides (or you can use another coverglass). 1 inch x 3 inch, about 1.2 mm thick.
- 3M double-stick tape (like the kind you can get at Office Max).
Anti-dig
Polyclonal sheep anti-dig from Roche (Cat. No. 1 333 089). This is shipped as a lyophilized powder. We always resuspend entire 200 microgram bottle with 1 ml of ice-cold PBS, and then make 20 microliter aliquots which are stored at -80C. An aliquot can be extracted from freezer, and diluted with 180 microliters of cold PBS to make:
20 microgram / milliliter working solution of anti-dig keep cold, store at +4C for up to a few weeks, or until you run out, or until stuff stops working
Procedure
Create a sample chamber
Details later (it's tough to describe, but easy to learn from someone)
- Clean glass
- Make chambers
- We find that making a bunch of chambers ahead of time doesn't save that much time, and they may not stay clean, and may losing sticking strength. So we make them on demand.
Form tethers
This assumes you have a typical sample chamber made out of #1 coverglass, double stick tape, and microscope slide (or another coverglass). Assumes sample volume about 10-20 microliters. Assumes glass is already cleaned, and dry.
- Coat the glass surfaces (and presumably the sticky tape walls) with anti-dig
- Because the sample is dry (and clean/hydrophilic)
